Binding assay using binding agents with tail groups

ABSTRACT

The present invention discloses methods and kits for the determination of the concentration of one or more analytes in a liquid sample using capture agents immobilized on a solid support and binding agents for binding the analyte(s), the binding agents having tail groups capable of binding to the respective capture agent. Preferably, the capture agents and binding agents are complementary oligonucleotides, and the capture agents are immobilized in the form of microspots. The use of the tail groups and capture agents can allow the binding of the analyte(s) to the binding agent(s) to take place in solution, rather than at a surface, improving the kinetics associated with this process. In addition, the user of the assay can customize any suitable binding agents for use with a universal support, by attaching tail groups to them.

FIELD OF THE INVENTION

The present invention relates to binding assays using binding agentswith tail groups, and in particular binding agents havingoligonucleotide tail groups. These binding assays are useful indetermining the concentration of analytes in liquid samples.

BACKGROUND OF THE INVENTION

It is known to measure the concentration of an analyte, such as a drugor hormone, in a liquid sample by contacting the liquid sample with abinding agent immobilised on a solid support, the binding agent havingbinding sites specific for the analyte, separating the binding agenthaving analyte bound to it and measuring a value representative of thefraction of the binding sites of the binding agent that are occupied bythe analyte. Typically, the concentration of the analyte in the liquidsample can then be determined by comparing the value representative ofthe fraction of the binding sites occupied by analyte against valuesobtained from a series of standard solutions containing knownconcentrations of analyte.

In the past, the measurement of the fraction of the binding sitesoccupied has usually been carried out by back-titration with a labelleddeveloping reagent using either so-called competitive or non-competitivemethods.

In the competitive method, the binding agent having analyte bound to itis back-titrated, either simultaneously or sequentially, with a labelleddeveloping agent, which is typically a labelled version of the analyteor an anti-idiotypic antibody capable of recognising empty binding sitesof the binding agent. The developing agent can be said to compete forthe binding sites on the binding agent with the analyte whoseconcentration is being measured.

The fraction of the binding sites which become occupied with thelabelled analyte can then be related to the concentration of the analyteas described above.

In the non-competitive method, the binding agent having analyte bound toit is back-titrated with a labelled developing agent capable of bindingto either the bound analyte or to the occupied binding sites on thebinding agent. The fraction of the binding sites occupied by analyte canthen be measured by detecting the presence of the labelled developingagent and, just as with competitive assays, related to the concentrationof the analyte in the liquid sample as described above.

In both competitive and non-competitive methods, the developing agent islabelled with a marker to allow the developing agent to be detected. Avariety of markers have been used in the past, for example radioactiveisotopes, enzymes, chemiluminescent markers and fluorescent markers.

In the field of immunoassay, competitive assays have in general beencarried out in accordance with design principles enunciated by Bersonand Yalow, for instance in “Methods in Investigative and DiagnosticEndocrinology” (1973), pages 111-116. Berson and Yalow proposed that inthe performance of competitive immunoassays, maximum sensitivity isachieved when an amount of binding agent is used to bind approximately30-50% of a low concentration of the analyte to be detected. Innon-competitive immunoassays, maximum sensitivity is generally thoughtto be achieved by using sufficient binding agent to bind close to 100%of the analyte in the liquid sample. However, in both cases immunoassaysdesigned in accordance with these widely-accepted precepts require thevolume of the sample to be known and the amount of binding agent used tobe accurately known or known to be constant.

In International Patent Application WO 84/01031, I disclosed that theconcentration of an analyte in a liquid sample can be measured bycontacting the liquid sample with a small amount of binding agent havingbinding sites specific for the analyte. In this “ambient analyte”method, provided the amount of binding agent is small enough to haveonly an insignificant effect on the concentration of the analyte in theliquid sample, it is found that the fraction of the binding sites on thebinding agent occupied by the analyte is effectively independent of thevolume of the sample.

This approach is further refined in EP 304,202 which discloses that thesensitivity and ease of development in the assays in WO 84/01031 areimproved by using an amount of binding agent less than 0.1V/K moleslocated on a small area (or “microspot”) on a solid support, where V isthe violume of the sample and K is the affinity constant of the bindingagent for the analyte. In both of these references, the fraction of thebinding sites occupied by the analyte is measured using either acompetitive or non-competitive technique as described above.

SUMMARY OF THE INVENTION

There is continuing need to develop binding assays which have enhancedkinetics to allow assays to be carried out more quickly and easily. Inaddition, it would be desirable to provide a binding assay which theuser of the assay can easily customise for the detection of differentgroups of analytes.

Accordingly, in a first aspect, the present invention provides a methodof determining the concentrations of analytes in a liquid samplecomprising:

(a) immobilising one or more capture agents on a solid support, eachcapture agent being capable of specifically binding a given bindingagent;

(b) contacting the liquid sample with one or more binding agents, eachbinding agent having binding sites specific for a given analyte so thata fraction of the binding sites become occupied by the analyte, and atail group adapted to bind to a corresponding capture agent;

(c) contacting the liquid sample, either simultaneously or sequentiallywith the step (b), with the immobilised capture agents so that thebinding agents become bound to their respective capture agents; and

(d) determining the fraction of the binding sites of a binding agentoccupied by analyte to determine the concentration of the analyte in theliquid samples.

Accordingly, the present invention provides an assay in which thebinding of the analytes takes place in the liquid phase, rather than ata surface of a solid substrate. This enhances the kinetics of thereaction between analyte and binding agent.

Thus, in one embodiment, contacting the liquid sample with the bindingand capture agents simultaneously allows the assay to be carried out ina single step, eg using a single reaction vessel. Alternatively,sequential contact of the binding agent(s), and capture agent(s) may bepreferred, especially where the liquid is serum or blood, andnon-specific binding is an important source of error. In these cases,the binding agent can be first contacted with the liquid sample in afirst vessel and then the sample transferred to a second vessel to allowthe capture agent to bind the binding agent to the solid support.

In a second aspect, the present invention provides a method ofimmobilising one or more binding agents on a support, each binding agenthaving binding sites specific for a given analyte and a tail groupadapted to bind to a capture agent, comprising:

(a) immobilising one or more capture agents on a support each captureagent being capable of binding to the tail group of a given bindingagent and,

(b) contacting the binding agents with the support having the captureagents immobilised thereon so that the binding agents becomespecifically bound to their respective capture agents through their tailgroups.

The above method can additionally comprise the step of attaching thetail groups to the binding agents prior to exposing them to the captureagents immobilised on the support.

Thus, it is possible for the user of the assay to customise bindingagents for use in determining the concentration of different groups ofanalytes and using the customised binding agents in conjunction with auniversal support having capture agents immobilised on it, to which thebinding agents can individually bind by virtue of their tail groups.

In this aspect of the invention, the assay is carried out by exposingthe support to a liquid sample after the binding agent(s) has or havebecome bound to the capture agent(s).

In either aspect, the present invention provides an assay in which thebinding agent is indirectly linked to capture agent immobilised on thesubstrate via the tail group.

Preferably, the capture agent is an oligonucleotide sequence which canhybridise to a complementary sequence comprising the tail group of thebinding agent. The oligonucleotides acting as capture agent or tail ofthe binding agent are sufficiently long to provide strong and specifichybridisation under the stringency conditions used in the assay.Typically, complementary oligonucleotides of at least about 8 or 9nucleotides in length are used. In a preferred embodiment, theoligonucleotides are preferably between 8 and 30 bases, more preferablybetween 16 and 20 bases, in length. However, the use of very longpolynucleotides is not preferred as these can lead to a reduction in thespecificity of binding different capture agents or to self hybridise,forming hairpin loops (double stranded regions). However, a suitablelength and sequence of oligonucleotide for a set of assay conditions canreadily be determined by those skilled in the art.

Conveniently, the binding agent is an antibody having binding sitesspecific for an analyte. Accordingly, when the capture agent on thesupport is exposed to the liquid phase binding agent, the binding agentbecomes bound to the solid support. Alternatively, where the analyte isa nucleic acid sequence, the binding agent can be an oligonucleotide.Thus, in this embodiment, the binding agent has a first sequence capableof hybridising to the analyte and a second sequence acting as the tailgroup.

Preferably, a small amount of binding agent is used in accordance withthe assays disclosed in WO 84/01031 or EP 304,202, so that the volume ofthe liquid sample need not be known. Thus, the amount of binding agentshould be sufficiently small so that it does not significantly affectthe ambient concentration of the analyte in the liquid sample.Typically, the use of an amount of binding agent which binds less than5% of the analyte is preferred. However, the use of a smaller amount ofbinding agent, eg to bind 2% or 1% of the analyte, further reduces thedisturbance to the ambient concentration of the analyte and helps tominimise the error in determining the analyte concentration.

Where the assay is conducted in accordance with EP 304,202 using lessthan 0.1V/K moles of binding agent, the affinity constant (K) for thebinding of analyte to binding agent is measured in accordance withnormal practice. This means the value of the affinity constant used todetermine how much binding agent constitutes 0.1V/K moles is the valuethat is obtained under the conditions (eg reactants, time of incubation,pH, temperature etc) that are used in the assay.

Preferably, each capture agent is used in excess to bind substantiallyall of a given binding agent. This maximises the assay sensitivity andensures that when the amount of binding agent used needs to be known orknown to be constant, the user of the assay can be confident thatsubstantially all of a binding agent used in an assay becomes bound toits capture agent on the support.

Preferably, molecules of capture agent are immobilised on a support atdiscrete locations, eg as microspots. This allows the concentration of aplurality of different analytes to be simultaneously determined using aplurality of different capture agents at a series of locations on thesupport. Where the capture agent(s) is or are immobilised as microspots,the sensitivity of the assay can be improved immobilising the captureagent at high density, thereby improving the signal-to-noise ratio (seefor example our co-pending application PCT/GB94/02814). Assuming samplevolumes of the order of 0.1-1.0 ml, the microspots preferably have anarea less than 1 mm² and a final surface density of binding agentbetween 1000 and 100000 molecules/μm².

Alternatively, a given capture agent can be immobilised on a support ata plurality of locations so that a series of measurements of theconcentration of an analyte can be made simultaneously.

Preferably, the fraction of the binding sites occupied by the analyte isdetected using developing agents in a competitive and/or non-competitivemethod as described above. The developing agents are capable of bindingto occupied or unoccupied binding sites of the binding agent or to boundanalyte and are labelled to enable bound developing agent to bedetected. Preferably, the developing agents are labelled antibodies.

The markers can be radioactive isotopes, enzymes, chemiluminescentmarkers or fluorescent markers. The use of fluorescent dye markers isespecially preferred as the fluorescent dyes can be selected to providefluorescence of an appropriate colour range (excitation and emissionwavelength) for detection. Fluorescent dyes include coumarin,fluorescein, rhodamine and Texas Red. Fluorescent dye molecules havingprolonged fluorescent periods can be used, thereby allowingtime-resolved fluorescence to be used to measure the strength of thefluorescent signal after background fluorescence has decayed. Latexmicrospheres containing fluorescent or other markers, or bearing them ontheir surface can also be employed in this context. The signals from themarkers can be measured using a laser scanning confocal microscope.

Alternatively, other high specific activity labels such aschemiluminescent labels can be used. In the case of chemiluminescentlabels, the signals from different chemilumiscent labels used to markbinding agent or developing agent can be simultaneously detected using,for example a charge-coupled device (CCD).

The binding agent (or a proportion of it) can conveniently be labelled,eg with a fluorophor. In accordance with the method set out in EP271,974, this means that it is not necessary for the user of the assayto know the amount of binding agent or to know that it is constant. Thisis because the ratio of the signals from the binding agent and thesignal indicating the fraction of the binding sites of the binding agentoccupied by analyte is dependent on the fraction of the sites of thebinding agent occupied by the analyte, but is independent of the totalamount of binding agent present.

Alternatively, if the user of the assay knows the volume of the sample,a larger amount of binding agent can be used so that the assay is notoperating under ambient analyte conditions. This allows theconcentration of the analyte to be determined using one label on thedeveloping agent and either knowing the amount of binding agent isconstant or labelling it with a second marker so that the amount isknown.

In a variant of this approach (described in our co-pending applicationPCT/GB94/02813), two labelled developing agents can be used, a firstcapable of specifically binding to unoccupied binding sites of thebinding agent and a second capable of binding to occupied binding sitesor bound analyte. Thus, the signal from either marker is representativeof the fraction of the binding sites occupied by analyte, while the sumof the signals is representative of the total amount of binding agentused.

This method can also avoid the necessity of knowing that a constantamount of binding agent is used as variations in the amount of bindingagent immobilised can readily be corrected for. Under thesecircumstances, the sample volume v must either be known or constant.This can be seen from the following formula show how the signals fromtwo labelled developing agents relates to the concentration of analytein a sample.

Let the signal emitted by the label marking the developing agentdirected against occupied binding agent binding sites be given by S_(o),

and the signal emitted by the label marking the developing agentdirected against unoccupied binding agent binding sites be given byS_(u),

and let the constants relating the respective signals to occupied andunoccupied sites be ε_(o) and ε_(u) respectively, and K=the effectiveequilibrium constant governing the reaction between the analyte andbinding agent.

Then, if the analyte concentration in a sample is given by Y,

Y=(S _(o)/ε_(o))[ε_(u)/(KS _(u))+1/v]

Assuming v is known, this equation contains two unknown constants, ε_(o)and ε_(u)/K. By determining the signals S_(o) and S_(u) for a series ofknown analyte concentrations, these constants can be determined, andunknown analyte concentrations estimated from correspondingdeterminations of S_(o) and S_(u). Thus, the assay need not work underambient analyte conditions.

Under ambient analyte conditions, the term 1/v becomes negligible, andS_(o)/S_(u) is proportional to the ambient analyte concentration.

In a first kit aspect, the present invention provides a kit fordetermining the concentrations of one or more analytes in a liquidsample in a method as described above, the kit comprising:

(a) a solid substrate having attached thereto at a plurality oflocations capture agent capable of specifically binding a binding agent;

(b) one or more binding agents, each binding agent having binding sitesspecific for an analyte, and a tail group adapted to bind one or morecapture agents; and

(c) one or more developing agents having markers capable of binding tooccupied binding agent binding sites or analyte bound to binding agentor unoccupied binding agent binding sites.

In a second kit aspect, the present invention provides a kit forcustomising an assay for the determination of the concentration of oneor more analytes comprising:

(a) one or more tail groups, each tail group being for attachment to abinding agent;

(b) a solid substrate having attached thereto at a plurality oflocations one or more capture agents capable of specifically binding toa tail group;

wherein the user of the assay attaches the tail groups to the bindingagents, thereby providing binding agents which can be used inconjunction with the solid substrate to which the capture agents areattached in a method as described above.

DESCRIPTION OF THE DRAWINGS

A preferred embodiment of the present invention will now be describedwith reference to the accompanying schematic drawings in which:

FIG. 1 shows an assay to detect two analytes in a liquid sample usingtwo species of capture agent and two species of binding agent, thecapture agent immobilised at two microspots;

FIG. 2 shows the assay of FIG. 1 in which the capture agent has becomebound to the binding agent;

FIG. 3 shows a non-competitive method of determining the occupancy ofthe binding agent using a second labelled antibody; and,

FIG. 4 shows a graph of signal plotted against TSH concentration fromthe experimental example below.

DETAILED DESCRIPTION

FIGS. 1 to 3 show a binding assay in which two species of binding agent2,4 having binding sites specific for different analytes 6,8 are used.Each binding agent 2,4 comprises an antibody 10,14 provided with anoligonucleotide tail group 12,16. The oligonucleotide tail groups havedifferent nucleotide sequences, the sequences being complementary to oneof the sequences of capture agents 18,20 immobilised on a solid support22 in the form of microspots. In this example, the oligonucleotides are8 nucleotides long.

In the assay, the two analytes 6,8 in the sample are exposed to bindingagents 2,4 so that a fraction of the analytes 6,8 become bound to theantibodies 10,14. As this reaction occurs in the liquid phase, thekinetics of the reaction between the antibodies 10,14 and the analytes(antigens) 6,8 are optimised.

Simultaneously or sequentially with the initial antibody/analytereaction, the liquid sample and binding agent are exposed to the solidsupport 22 having capture agents 18,20 immobilised on it. This allowsthe nucleotide sequences 12,16 of the binding agents 2,4 to bind to thecomplementary sequences of the capture agents 18,20 immobilised on thesupport 22. This is shown in FIG. 2. However, the capture agents 16,18are generally used in excess to ensure that substantially all thebinding agent 10,14 is bound to the support 22. Thus, in FIGS. 2 and 3,one molecule of capture agent 28 is left unoccupied. The fraction of thebinding sites of the binding agents 2,4 can then be determined using aconventional back-titration technique. Thus, in FIG. 3 labelledantibodies 24,26 are used in a non-competitive technique to mark thepresence of occupied binding agents 2,4 respectively. As the antibodies24,26 are labelled with markers (not shown) a fraction of the bindingsites of the binding agents 2,4 can then be determined. This in turnallows the concentration of the analytes in the liquid sample to befound, eg by reference to results obtained using a series of solutionsof known analyte concentration.

The assay shown in FIGS. 1 to 3 can be adapted to measure theconcentration of any pair of analytes using the same solid support 22having capture agents 18,20 immobilised on it. This can be done byproviding binding agent suitable for binding an analyte with anoligonucleotide tail group 12,16 so that the binding agents willspecifically bind to one of the microspots 18,20. Thus, it is envisagedthat the user of the assay will be able to customise his or her bindingagent for use with a universal array of microspots.

EXAMPLE Reagents

1) Mouse IgG (monoclonal anti-TSH) from the Scottish Antibody ProductionUnit (SAPU).

2) Rabbit IgG, goat anti-mouse IgG (whole molecule) and goat anti-rabbitIgG (whole molecule) antibodies from Sigma.

3) Sulfate Fluospheres, 0.1 μm diameter, yellow/green fluorescent (ex490; em 515 nm) and Sulfate Fluospheres, 0.1 μm diameter, redfluorescent (ex 580; em 605 nm) from Molecular Probes.

4) Oligonucleotides from Oswell DNA Service:

a) CACACACACACACACACA with 5′-biotin modification (poly-CA)

b) GTGTGTGTGTGTGTGTGT with 51′-phosphorothioate modification (poly-GT)

c) GAGAGAGAGAGAGAGAGA with 5′-biotin modification (poly-GA)

d) CTCTCTCTCTCTCTCTCT with 5′-phosphorothioate modification (poly-CT)

5) Sulfo-LC-SPDP {sulfosuccinimidyl6-[3′-(2-pyridyldithio)-propionamido]hexanoate} from Pierce.

6) PD10 columns and Sephadex G200 from Pharmacia.

7) RIA grade Bovine Serum Albumin (BSA), Tween20, sodium azide,di-sodium hydrogen orthophosphate anhydrous, sodium di-hydrogenorthophosphate, EDTA and Trizma from Sigma

8) Avidin DX from Vector Laboratories

9) Centricon-30 and Centriprep-30 concentrators from Amicon

10) Thyroid stimulating hormone (TSH) from NIH USA

Adsorption of Anti-Mouse IgG and Anti-Rabbit IgG Antibodies to SulfateFluoSpheres

1) A 0.5 ml aliquot of 2% (10 mg), 0.1 μm yellow/green FluoSpheres wasadded to 2 mg of goat anti-mouse IgG antibody dissolved in 0.5 ml 0.1 μMphosphate buffer, pH7.4. A 0.5 ml aliquot of 2% (10 mg), 0.1 μm redFluoSpheres was added to 2 mg of goat anti-rabbit IgG antibody dissolvedin 0.5 ml 0.1M phosphate buffer, pH7.4. Both preparations were shakenovernight at room temperature.

2) The two preparations were centrifuged for 10 min at 8° C. in a MSEHigh-Spin 21 Ultra-centrifuge.

3) Each pellet was dispersed in 2 ml of 1% BSA in phosphate buffer,shaken for 1 hour at room temperature and centrifuged as above.

4) Each pellet was dispersed in 2 ml of 0.5% Tween20 in phosphatebuffer, shaken for 30 min at room temperature and centrifuged as above.

5) Each pellet was dispersed in 2 ml of phosphate buffer and centrifugedas above.

6) Each pellet was dispersed in 2 ml of phosphate buffer and centrifugedas above.

7) Each pellet was dispersed in 2 ml of 1% BSA containing 0.1% sodiumazide and stored at 4° C.

Conjugation of Mouse Monoclonal IgG and Rabbit IgG to Oligonucleotides

1) 3 mg of sulpho-LC-SPDP was added to 4.6 mg of mouse anti-TSHmonoclonal or rabbit IgG dissolved in 1 ml of PBS/EDTA and shaken for 30min at room temperature.

2) The activated antibodies were separated from unreacted SPDP on PD10columns. The samples were eluted with PBS/EDTA and 0.5 ml fractionscollected.

3) The fractions from the first peak containing the activated antibodywere pooled and concentrated using a Centricon-30 concentrator toapproximately 10 μl.

4) 100 nM of 5′-phosphorothioate modified poly-GT oligonucleotide wasadded to 14.8 nM of the activated mouse monoclonal IgG. 58.3 nM of5′-phosphorothioate modified poly-CT oligonucleotide was added to 8.7 nMof the activated rabbit IgG. Both preparations were made up to 1 ml withPBS/EDTA and shaken overnight at room temperature.

5) The oligonucleotide conjugated mouse and rabbit IgG preparations wereseparated from unreacted oligonucleotides on a Sephadex G200 column(1.5×45 cm). The samples were eluted with PBS/EDTA and 2 ml fractionscollected.

6) The fractions from the first peak containing the oligonucleotideconjugated antibody were pooled and concentrated using a Centriprep-30concentrator to approximately 500 μl and stored at 4° C.

To Demonstrate That a Mixture of Oligonucleotide-Conjugated AntibodiesWould Hybridize Only With Complementary Oligonucleotide Deposited on aSolid-Phase as Microspots

1) Dynatech black Microfluor microtitre wells were coated with 50 μl ofavidin-DX in 0.1M bicarbonate buffer, pH 8.5 and at a concentration of 5μg/ml for 5 min at room temperature.

2) After washing with 0.01M phosphate buffer, the avidin coatedmicrotitre wells were blocked with 200 μl of 1% BSA for 1 hour at roomtemperature and washed again with the same buffer and dried.

3) A 0.25 μl droplet of each of the two 5′-biotin modified poly-CA andpoly-GA oligonucleotides in 0.1% BSA and at a concentration of 0.025nM/ml were deposited on opposite sides of avidin coated microtitre wellsand allowed to react for 30 min under a moist atmosphere. The dropletswere then aspirated and the microtitre wells washed with phosphatebuffer.

4) A 50 μl aliquot of Tris-HCI assay buffer containing 0.25 μg/ml eachof the poly-GT-conjugated mouse monoclonal IgG and poly-CT-conjugatedrabbit IgG was added to all but the control microtitre wells (50 μl ofassay buffer containing unconjugated mouse and rabbit IgG was added tothe control wells instead), shaken for 1 hour under a moist atmosphereand washed with phosphate buffer containing 0.05% Tween20.

5) A 200 μl aliquot of Tris-HCI assay buffer containing 0.3 μg/ml goatanti-mouse IgG antibody conjugated yellow/green FluoSpheres and 0.6pg/mlgoat anti-rabbit IgG antibody conjugated red FluoSpheres was added toall microtitre wells, shaken for 1 hour at room temperature, washed withphosphate-Tween20 buffer and scanned with a confocal laser scanningmicroscope equipped with an Argon/Krypton laser.

Results

Excitation: 488DF10

Emission: 525DF35

Yellow/Green Sample Signal Control 13.3 ± 0.5 Avidin- - -B-Poly-CA- --Poly-GT-Mouse 100.9 ± 10.9 IgG microspot Avidin- - -B-Poly-GA- --Poly-CT-Rabbit 16.9 ± 0.3 IgG microspot

Excitation: 568DF10

Emission: 585EFLP

Sample Red Signal Control 22.0 ± 0.2 Avidin- - -B-Poly-CA- --Poly-GT-Mouse 24.0 ± 0.4 IgG microspot Avidin- - -B-Poly-GA- --Poly-CT-Rabbit 99.8 ± 2.7 IgG microspot

Conclusions

(1) The poly-GT oligonucleotide tagged mouse IgG hybridized only withcomplementary biotinylated poly-CA but not non-complementarybiotinylated poly-GA oligonucleotide microspots deposited on the samemicrotitre well.

(2) The poly-CT oligonucleotide tagged rabbit IgG hybridized only withcomplementary biotinylated poly-GA but not non-complementarybiotinylated poly-CA oligonucleotide microspots deposited on the samemicrotitre well.

To Demonstrate Antigen Binding of the Oligonucleotide Tagged AntibodyMicrospots

1) Dynatech black Microfluor microtitre wells were coated with 50 μl ofavidin-DX in 0.1 M bicarbonate buffer, pH 8.5 and at a concentration of5 μg/ml for 5 min at room temperature.

2) After washing with 0.01M phosphate buffer, the avidin coatedmicrotitre wells were blocked with 200 μl of 1% BSA for 1 hour at roomtemperature and washed again with the same buffer and dried.

3) A 0.25 droplet of 5′-biotin modified poly-CA oligonucleotide in 0.1%BSA and at a concentration of 0.025nM/ml was deposited on each of theavidin coated microtitre wells and allowed to react for 30 min under amoist atmosphere. The droplets were then aspirated and the microtitrewells washed with phosphate buffer.

4) A 50 μl aliquot of Tris-HCI assay buffer containing 0.25 μg/ml of thepoly-GT-conjugated anti-TSH mouse monoclonal IgG was added to themicrotitre wells, shaken for 1 hour under a moist atmosphere and washedwith phosphate buffer containing 0.05% Tween2.

5) A 200 μl aliquot of TSH standards in Tris-HCI assay buffer (0, 0.1,0.3 & 1.0 μU/ml) was added to triplicate wells and incubated for 1 hourat room temperature and washed with phosphate-Tween20 buffer.

6) A 200 μl aliquot of 50 μg/ml anti-TSH developing antibody conjugatedyellow/green sulfate FluoSpheres was added to all microtitre wells,shaken for 1 hour at room temperature, washed with phosphate-Tween20buffer and scanned with a confocal laser scanning microscope equippedwith an Argon/Krypton laser.

RESULTS AND CONCLUSION

The poly-GT oligonucleotide tagged anti-TSH mouse monoclonal IgG wasfully functional as demonstrated by the successful generation of astandard curve when it was used as binding antibody deposited on thesolid-phase via biotinylated complementary poly-CA oligonucleotidecoupled to avidin coated microtitre wells (see FIG. 4).

4 1 18 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide 1 cacacacaca cacacaca 18 2 18 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide 2 gtgtgtgtgt gtgtgtgt18 3 18 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide 3 gagagagaga gagagaga 18 4 18 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide 4 ctctctctct ctctctct18

What is claimed is:
 1. A method for determining the concentration of aplurality of analytes in a liquid sample, the method employing: a solidsupport having immobilized at discrete locations thereon a plurality ofdifferent capture agents; and a plurality of binding agents, eachbinding agent having binding sites specific for a given analyte andhaving a tail group adapted to bind to one of said capture agents; themethod comprising the steps of: (a) contacting the liquid sample withthe binding agents so that a fraction of the binding sites of eachbinding agent become occupied by their respective analytes; (b)contacting the liquid sample, either simultaneously or sequentially withstep (a), with the solid support so that the binding agents become boundto their respective capture agents; and (c) determining a valuerepresentative of the fraction of the binding sites of a given bindingagent occupied by an analyte whereby to determine the concentration ofthe analyte in the liquid sample wherein the capture agents areoligonucleotides having sequences which can hybridize to a complementarysequence on the tail group of the corresponding binding agent.
 2. Amethod according to claim 1 wherein the oligonucleotides are between 8and 30 bases long.
 3. A method according to claim 1 wherein the bindingagent is an antibody having binding sites specific for an analyte.
 4. Amethod according to claim 1 wherein a small amount of each binding agentis used so that the ambient concentration of the analyte for which thebinding agent is specific is not significantly disturbed.
 5. A methodaccording to claim 4 wherein the small amount of binding agent is lessthan 0.1V/K moles, where V is the volume of the sample and K is theeffective affinity constant for the analyte binding to the bindingagent.
 6. A method according to claim 1 wherein each capture agent isused in excess to bind substantially all of a given binding agent.
 7. Amethod according to claim 1 wherein the discrete locations aremicrospots.
 8. A method according to claim 1 wherein a given captureagent is immobilized on the support at a plurality of locations so thata series of measurements of the concentration of a given analyte can bemade simultaneously.
 9. A method according to claim 1 wherein the valuerepresentative of the fraction of the binding sites occupied by theanalyte is determined using developing agents in a competitive and/ornon-competitive method, the developing agents being labelled withmarkers.
 10. A method according to claim 9 wherein the marker arefluorescent or chemiluminescent markers.
 11. A kit for determining theconcentrations of a plurality of analytes in a liquid sample in a methodaccording to claim 1, the kit comprising: (a) a solid substrate havingattached thereto at discrete locations a plurality of different captureagents capable of specifically binding a given binding agent; (b) aplurality of binding agents, each binding agent having binding sitesspecific for a given analyte, and having a tail group adapted to bind ofsaid capture agents; and (c) one or more developing agents havingmarkers capable of binding to occupied binding agent binding sites oranalyte bound to binding agent or unoccupied binding agent bindingsites; wherein the capture agents are oligonucleotides having sequenceswhich can hybridize to a complementary sequence on the tail group of thecorresponding binding agent.
 12. A kit for customizing an assay for thedetermination of the concentration of one or more analytes in a liquidsample, the kit comprising: (a) one or more tail groups, each tail groupbeing for attachment to a binding agent; (b) a solid substrate havingattached thereto at discrete locations a plurality of different captureagents capable of specifically binding to a tail group; wherein thecapture agents are oligonucleotides having sequences which can hybridizeto a complementary sequence on the tail group of the correspondingbinding agent and the user of the assay attaches the tail groups to thebinding agents, thereby providing binding agents which can be used inconjunction with the solid substrate to which the capture agents areattached in a method according to claim
 1. 13. The method of claim 1further comprising the initial step of immobilizing the plurality ofdifferent capture agents on the solid support at the discrete locations.14. A method for determining a value representative of a fraction ofbinding sites of a binding agent, having binding sites specific for ananalyte, that are occupied by at least one analyte present in a liquidsample, the method employing: a solid support having immobilized atdiscrete locations thereon a plurality of different capture agents; anda plurality of binding agents, each binding agent having binding sitesspecific for a given analyte and having a tail group adapted to bind toone of said capture agents, the method comprising the steps of: (a)contacting the liquid sample with the binding agents, so that a fractionof the binding sites of each binding agent become occupied by theirrespective analytes; (b) contacting the liquid sample, eithersimultaneously or sequentially with step (a), with the solid support sothat the binding agents become bound to their respective capture agents;and (d) determining a value representative of the fraction of thebinding sites of a given binding agent occupied by an analyte, whereinthe capture agents are oligonucleotides having sequences that hybridizeto a complementary sequence on the tail group of the correspondingbinding agent.
 15. A method according to claim 14, wherein the valuerepresentative of the fraction of the binding sites occupied by ananalyte is determined using developing agents in a method selected fromat least one of the group consisting of a competitive method and anon-competitive method, said developing agents being labelled withmarkers.